Controlled partial restriction digestions of DNA by competition with modification methyltransferases

Competitive reactions, using defined ratios of DNA restriction methyltransferase to endonuclease, are shown to result in reliable partial restriction digests of DNA. This method is suitable over a wide range of DNA concentrations and works on DNA in liquid or embedded in agarose. Simultaneous methylase/endonuclease reactions using endonucleases that cleave human DNA very infrequently, such as ClaI or NotI, should generate very large discrete partial DNA fragments suitable for physical mapping in the million base-pair range. Another possible application of methylase/endonuclease competitive reactions is the production of defined partial digests for making cosmid, lambda, or other genomic libraries.     

Methylase-limited partial NotI cleavage for physical mapping of genomic DNA.

Partial cleavage of DNA with the restriction endonuclease NotI (5'...GC/GGCCGC...3') is an important technique for genomic mapping. However, partial genomic cleavage with this enzyme is impaired by the agarose matrix in which the DNA must be suspended. To solve this problem we have purified the blocking methylase M. BspRI (5'...GGmCC...3') for competition digests with NotI. The resulting methylase-limited partial DNA cleavage is shown to be superior to standard techniques on bacterial genomic DNA. Abbreviations: bp, base-pair; kb, one thousand base-pairs; Mb, one million base-pairs; Tris, Tris(hydroxy-methyl)aminomethane; EDTA, (ethylenedinitrilo)tetraacetic; beta-ME, beta-mercaptoethanol; PMSF, phenyl methyl-sulfonyl fluoride; PEG, polyethyleneglycol (MW = 8000); 3H, tritium; SAM, S-adenosylmethionine; KGB, potassium glutamate buffer; DTT, dithiothreitol; IPTG, isopropyl-beta-D-thiogalactopyranoside; BSA, bovine serum albumin.     

Scattering of the rRNA genes on the physical map of the circular chromosome of Leptospira interrogans serovar icterohaemorrhagiae.

Leptospira interrogans is a pathogenic bacterium with a low G+C content (34 to 39%). The restriction enzymes NotI, AscI, and SrfI cut the chromosome of L. interrogans serovar icterohaemorrhagiae into 13, 3, and 5 fragments separable by one- and two-dimensional pulsed-field gel electrophoresis (PFGE). The genome is composed of a circular 4.6-Mbp chromosome and a 0.35-Mbp extrachromosomal element. A physical map of the chromosome was constructed for NotI, AscI, and SrfI by using single and double digests, or partial NotI digests obtained at random or by cross-protection of NotI sites by FnuDII methylase, and linking clones. rRNA genes were found to be widely scattered on the chromosome.     

Control of partial digestion combining the enzymes dam methylase and MboI.

A method is described which allows the preparation of reproducible partial digests without previous establishment of the incubation conditions. It is based on a combined application of dam methylase and the restriction endonuclease MboI, both recognizing the sequence 5'-GATC-3' but MboI unable to cut the methylated site. Due to their competition for the same substrate the DNA is partially digested, with the size of the resulting fragments strongly dependent on the ratio of enzymes. The Km of the dam methylase was determined to be 115 ng DNA/microliters indicating a variance in fragment sizes generated at low DNA-concentrations. This effect is minimized above 150 ng/microliters. Any influence of digestion time is avoided, because the reaction runs until complete modification of all sites. The dependence on enzyme concentration and presence of agarose was checked. Knowledge of these parameters allows an accurate prediction of fragment sizes generated at different conditions. The technique was successfully used to construct libraries from different sources, in particular chromosome-specific libraries from small amounts of flow-sorted material.     

Isolation and Characterization of Site-Specific DNA-methyltransferases from Bacillus coagulans K

Two site-specific DNA methyltransferases, M.BcoKIA and M.BcoKIB, were isolated from the thermophilic strain Bacillus coagulans K. Each of the methylases protects the recognition site 5'-CTCTTC-3'/5'-GAAGAG-3' from cleavage with the cognate restriction endonuclease BcoKI. It is shown that M.BcoKIB is an N6-adenine specific methylase and M.BcoKIA is an N4-cytosine specific methylase. According to bisulfite mapping, M.BcoKIA methylates the first cytosine in the sequence 5'-CTCTTC-3'.     

Organization of delta-crystallin genes in the chicken.

Double-stranded DNA was synthesized from delta-crystallin mRNA prepared from lens fibers of 15-day-old chick embryos and cloned at the Pst I site of the plasmid pBR322. Using the cloned cDNA and single-stranded cDNA as hybridization probes, a number of genomic DNA fragments containing delta-crystallin gene sequences have been cloned from the partial and complete EcoRI digests of chick brain DNA. One of the clones from the partial digests contains a DNA fragment that consists of four EcoRI fragments of 7.6 kb, 4.0 kb, 2.6 kb, and 0.8 kb. The gene sequences reside in the (5')7.6 kb - 0.8 kb - 4.0 kb (3') fragments. Electron microscopy has provided evidence that the cloned DNA fragment includes the entire gene sequences complementary to delta-crystallin mRNA except for the 3' terminal poly(A) tail, and that the delta-crystallin gene is interrupted by at least 13 intervening sequences. Another clone contains a genomic fragment that consists of two EcoRI fragments of 3.0 kb and 11 kb. The DNA fragment in the latter clone represents a different delta-crystallin gene, as judged by restriction endonuclease mapping and by electron microscopy.     

Toward a long-range map of human chromosomal band 22q11

Human chromosome band 22q11 is involved in numerous chromosomal rearrangements. A long-range molecular map of this region would allow the more precise localization of the various breakpoints of these rearrangements. Toward this goal we have constructed a genomic DNA library that allows the isolation of DNA clones that are directly adjacent to NotI sites. NotI was chosen because it is a restriction enzyme that digests infrequently in the human genome. The genomic DNA used in this library was from a human/hamster hybrid cell line that has a chromosome 22 as the only visible human chromosome. Two clones were isolated and mapped to different regions of 22q11 using a somatic cell hybrid mapping panel. A long-range restriction map flanking the NotI site of each of these two clones was produced using NotI and other infrequently cutting enzymes. Both NotI sites analyzed were located in HTF islands, regions often associated with the 5' end of genes. Thus, the NotI map of 22q11 may also aid in the cloning of undiscovered genes, giving a starting point for the study of duplication/deficiency syndromes of the region.     

A column method for determination of DNA cytosine-C5-methyltransferase activity

DNA methylation at the 5th position of cytosine has been found to be correlated with tumorigenesis. An inhibitor of DNA methylase could, therefore, be used as an anticancer drug. However, only a few inhibitory compounds have been discovered due to the limitations for assaying the DNA methylation. In this study, we describe a modification of DNA cytosine-C5-methyltransferase assay system utilizing [(3)H]-labeled S-adenosyl-methionine (SAM) and Sephadex G-25 column. Pre-treatment of either lambda DNA or the promoter region of human telomerase (hTERT) with HaeIII methylase greatly reduced the digestion of the DNAs with the corresponding restriction enzyme HaeIII endonuclease (over 100-fold), and the result was further confirmed by agarose gel electrophoresis. Application of this column method to another modification/restriction system, EcoRI methylase/endonuclease, gave rise to the similar results. Our data suggest that the newly developed column method could be effective for rapid screening of large number of cytosine methylase inhibitors and could also be applicable to other DNA methylases.     

Structures of the rare-cutting restriction endonuclease NotI reveal a unique metal binding fold involved in DNA binding

The structure of the rare-cutting restriction endonuclease NotI, which recognizes the 8 bp target 5'-GCGGCCGC-3', has been solved with and without bound DNA. Because of its specificity (recognizing a site that occurs once per 65 kb), NotI is used to generate large genomic fragments and to map DNA methylation status. NotI contains a unique metal binding fold, found in a variety of putative endonucleases, occupied by an iron atom coordinated within a tetrahedral Cys4 motif. This domain positions nearby protein elements for DNA recognition, and serves a structural role. While recognition of the central six base pairs of the target is accomplished via a saturated hydrogen bond network typical of restriction enzymes, the most peripheral base pairs are engaged in a single direct contact in the major groove, reflecting reduced pressure to recognize those positions. NotI may represent an evolutionary intermediate between mobile endonucleases (which recognize longer target sites) and canonical restriction endonucleases.     

Shotgun polymerase chain reaction: construction of clone libraries specific to a NotI fragment of flow-sorted human chromosome 22.

We have developed the "shotgun polymerase chain reaction," a method for obtaining a large number of DNA markers specific to a giant DNA fragment, which facilitates analysis of a particular chromosomal region. We applied this method to a giant NotI fragment which carries the immunoglobulin lambda constant region on chromosome 22. NotI digests of chromosome 22 flow-sorted from human B-lymphoblastoid cell line GM130B were size fractionated by pulsed-field gel electrophoresis. Preliminary Southern hybridization analysis revealed that the immunoglobulin lambda constant region was conveyed on 1.4- and 1.3-Mb NotI fragments in this cell line. The agarose gel corresponding to 1.2 to 1.5 Mb in size was excised into slices and subjected to polymerase chain reaction to identify gel slices containing NotI fragments carrying Ke-Oz+, a subtype of the immunoglobulin lambda constant region. From the NotI fragment thus identified, a large number of small DNA segments were amplified through the ligation-mediated random polymerase chain reaction method. The amplified products were cloned and analyzed for chromosomal origin and localization to particular NotI fragments. Seven of eighteen clones originated from the 1.4-Mb NotI fragment of chromosome 22 in GM130B cells, which appears to be exactly the same as detected by a probe for the immunoglobulin lambda constant region.     

New cloning vectors and techniques for easy and rapid restriction mapping

We have modified plasmid, phage lambda and cosmid cloning vectors to be of general use for easily and unambiguously determining restriction maps of recombinant DNA molecules. Each vector is constructed so that it contains the rarely found NotI restriction site joined to a short synthetic linker sequence that is followed by a multiple cloning site. DNA cloned into these vectors may be restriction-mapped by either of two methods. In one technique, the cloned DNA is completely digested with NotI, followed by partial digestion with any other restriction enzyme. After electrophoresis and transfer to a nylon membrane, the fragments are hybridized to a labeled probe complementary to the NotI linker. In the second technique, referred to as recession hybridization detection, cloned DNA is digested with NotI and then briefly treated with exonuclease III to recess the 3' ends. After hybridizing a labeled complementary oligodeoxynucleotide to the single-stranded 5' end containing the linker sequence, the DNA is partially digested with another restriction enzyme, electrophoresed and the gel is exposed to x-ray film. With either method the size of each labeled fragment corresponds directly to the distance that a restriction site is located from the NotI linker terminus. Methods for obtaining partial restriction enzyme digests have been devised so that as many as 20 different enzymes may be conveniently mapped on a single gel in little more than a day. The vectors and techniques described may also be adapted to automated or semi-automated devices that read fragment lengths and calculate the resulting restriction map.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of Dpn I endonuclease.

The recognition sequence for the dam methylase of Escherichia coli K12 has been determined directly by use of in vivo methylated ColE1 DNA or DNA methylated in vitro with purified enzyme. The methylase recognizes the symmetric tetranucleotide d(pG-A-T-C) and introduces two methyl groups per site in duplex DNA with the product of methylation being 6-methylaminopurine. This work has also demonstrated that Dpn I restriction endonuclease cleaves on the 3' side of the modified adenine within the methylated sequence to yield DNA fragments possessing fully base-paired termini. All sequences in ColE1 DNA methylated by the dam enzyme are subject to double strand cleavage by Dpn I endonuclease. Therefore, this restriction enzyme can be employed for mapping the location of sequences possessing the dam modification.     

Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.     

Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.     

A comparison of vertebrate interferon gene families detected by hybridization with human interferon DNA

Cloned human interferon complementary DNAs were used as hybridization probes to detect interferon alpha and beta gene families in restriction endonuclease digests of total genomic DNA isolated from a wide range of vertebrates and invertebrates. A complex interferon-alpha multigene family was detected in all mammals examined, whereas there was little or no cross-hybridization of human interferon-alpha complementary DNA to non-mammalian vertebrates or invertebrates. In contrast, human interferon-beta complementary DNA detected one or two interferon-beta genes in all mammals tested, with the exception of the cow and the blackbuck, both of which possessed a complex interferon-beta multigene family which has presumably arisen by a recent series of gene duplications. Interferon-beta sequences could also be detected in non-mammalian vertebrates ranging from birds to bony fish. Detailed restriction endonuclease mapping of DNA sequences neighbouring the interferon-beta gene in a variety of primates indicated a strong evolutionary conservation of flanking sequences, particularly on the 3' side of the gene.     

Proteins encoded by the DpnII restriction gene cassette. Two methylases and an endonuclease

Proteins encoded by three genes in the DpnII restriction enzyme cassette of Streptococcus pneumoniae were purified and characterized. Large amounts of the proteins were produced by subcloning the cassette in an Escherichia coli expression system. All three proteins appear to be dimers composed of identical polypeptide subunits. One is the DpnII endonuclease, and the other two are DNA adenine methylase active at 5' GATC 3' sites. Inactivation of enzyme activity by insertions into the genes and comparison of the DNA sequence with the amino-terminal sequence of amino acid residues in the proteins demonstrated the following correspondence between genes and enzymes. The promoter-proximal gene in the operon, dpnM, encodes a 33 X 10(3) Mr polypeptide that gives rise to a potent DNA methylase. The next gene, dpnA, encodes the 31 x 10(3) Mr polypeptide of a weaker and less-specific methylase. The third gene, dpnB, encodes the 34 x 10(3) Mr polypeptide of the endonuclease. Although the endonuclease polypeptide is initiated from an ordinary ribosome-binding site, each of the methylase polypeptide begins at an atypical site with a consensus sequence entirely different from that of Shine & Dalgarno. This presumptive novel ribosome-binding site is well recognized in both S. pneumoniae and E. coli.     

Methylation of intact chromosomes by bacterial methylases in agarose plugs suitable for pulsed-field electrophoresis. Methylation of intact chromosomes in agarose by methylases

Conditions were determined for the methylation of intact yeast chromosomes by EcoRI, HhaI, and MspI bacterial methylases using an endonuclease protection assay while the chromosomes were embedded in agarose plugs suitable for transverse-field electrophoresis. Parameters were also established for the methylation of human chromosomes by EcoRI methylase. Methylation of embedded chromosomes by EcoRI methylase required prewashes with EDTA. EcoRI, HhaI, and MspI methylases showed optimal activity when nonacetylated bovine serum albumin, high levels of S-adenosylmethionine, and high levels of methylase were used. The use of bacterial methylases for methylation of embedded chromosomes will allow investigators to normalize variations in cellular DNA methylation prior to restriction and create new and rare endonuclease recognition sites which will facilitate the detection of chromosomal alterations and deletions.     

McrI: a novel class-II restriction endonuclease from Micrococcus cryophilus recognizing 5'-CGRY/CG-3'

A new class-II restriction endonuclease, McrI, with a novel sequence specificity as isolated from the Gram-positive eubacterium Micrococcus cryophilus. McrI recognizes the palindromic hexanucleotide sequence. [sequence: see text] The novel enzyme in the presence of Mg2(+)-ions cleaves specifically both strands as indicated by the arrows. The staggered cuts generate 3'-protruding ends with single-stranded 5'-RY-3' dinucleotide extensions. The McrI recognition sequence was deduced from mapping data on DNAs of bacteriophages theta X174RF and M13mp18RF characterized by one and four cleavage sites, respectively. The cut positions within both strands of the recognition sequence were determined in sequencing experiments by analyzing hydrolysis of phosphodiester bonds within a polylinker region of M13mp18RF DNA containing an additional McrI recognition site including treatment with T4 DNA polymerase. The novel enzyme may be a useful tool for cloning experiments by completion of the enzymes EclXI (5'-C/GGCCG-3'), NotI (5'-GC/GGCCGC-3'), PvuI (5'-CGAT/CG-3') as well as EaeI (5'-Y/GGCCR-3') and XhoII (5'-Y/GATCR-3') characterized by partly identical sequence specificities.     

Cleavage at the twelve-base-pair sequence 5''-TCTAGATCTAGA-3'' using M.Xbal (TCTAGm6A) methylation and DpnI (Gm6A/TC) cleavage.

The DNA methylase M.Xbal was isolated from an E. coli recombinant clone. We deduce that the enzyme methylates at the sequence 5'-TCTAGm6A-3'. In combination with the methylation-dependent restriction endonuclease, DpnI (5'-Gm6A/TC-3'), DNA cleavage occurs at the sequence 5'-TCTAGA/TCTAGA-3'. This twelve-base-pair site should occur once every 16,000,000 base pairs in a random sequence of DNA. The exceptional rarity of the M.XbaI/DpnI sequence makes it an ideal candidate for transpositional integration of a unique cleavage site into bacterial genomes. Retrotransposition into mammalian genomes is also an attractive possibility.